"Pseudo-pseudogenes" in bacterial genomes: Proteogenomics reveals a wide but low protein expression of pseudogenes in Salmonella enterica.

Pseudogenes (genes disrupted by frameshift or in-frame stop codons) are ubiquitously present in the bacterial genome and considered as nonfunctional fossil. Here, we used RNA-seq and mass-spectrometry technologies to measure the transcriptomes and proteomes of Salmonella enterica serovars Paratyphi A and Typhi. All pseudogenes' mRNA sequences remained disrupted, and were present at comparable levels to their intact homologs. At the protein level, however, 101 out of 161 pseudogenes suggested successful translation, with their low expression regardless of growth conditions, genetic background and pseudogenization causes. The majority of frameshifting detected was compensatory for -1 frameshift mutations. Readthrough of in-frame stop codons primarily involved UAG; and cytosine was the most frequent base adjacent to the codon. Using a fluorescence reporter system, fifteen pseudogenes were confirmed to express successfully in vivo in Escherichia coli. Expression of the intact copy of the fifteen pseudogenes in S. Typhi affected bacterial pathogenesis as revealed in human macrophage and epithelial cell infection models. The above findings suggest the need to revisit the nonstandard translation mechanism as well as the biological role of pseudogenes in the bacterial genome.

Constructing conjugate vaccine against Salmonella Typhimurium using lipid-A free lipopolysaccharide.

BACKGROUND: Salmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium. METHODS: To develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS-linker-protein conjugates 6a-9b. The designed bifunctional linkers 1-5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier. RESULTS: The synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol-sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS-linker-protein vaccines at a dosage of 2.5 µg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a-9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge. CONCLUSIONS: This study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein.

Disulfidptosis and ferroptosis related genes predict prognosis and personalize treatment for hepatocellular carcinoma.

Background: Understanding the interplay between disulfidptosis, ferroptosis, and hepatocellular carcinoma (HCC) could provide valuable insights into the pathogenesis of HCC and potentially identify novel therapeutic targets for the treatment of this deadly disease. This study aimed to identify a prognostic signature for HCC by examining the differential expression of genes related to disulfidptosis and ferroptosis (DRG-FRG), and to assess its clinical applicability. Methods: By integrating 23 disulfidptosis and 259 ferroptosis related genes with HCC messenger RNA (mRNA) expression data from The Cancer Genome Atlas (TCGA), differentially expressed DRG-FRG genes were identified. From these, 11 DRG-FRG genes were selected to construct a risk signature model using least absolute shrinkage and selection operator regression analyses. The prognostic performance of this model was evaluated by Kaplan-Meier survival analysis and time-dependent receiver operating characteristic (ROC) analysis. Subsequently, a nomogram was built by combining the signature with clinical variables. To further delve into the underlying mechanisms, we performed bioinformatics analysis using a variety of databases. Results: A prognostic signature based on 11 DRG-FRG genes effectively categorized HCC patients into high- and low-risk groups, showing a significant survival difference. Even after considering clinical variables, this signature remained an independent prognostic factor. Furthermore, the signature played a role in various critical biological processes and pathways that drive HCC progression. Potential therapeutic benefits could be derived from small molecule drugs targeting NQO1 and SLC7A11. Interestingly, the high-risk group exhibited resistance to several chemotherapeutic drugs, yet showed sensitivity to others when contrasted with the low-risk group. Lastly, the DRG-FRG genes signature had a strong correlation with the tumor immune microenvironment, marked by an elevated expression of immune checkpoint molecules in the high-risk group. Conclusions: The signature based on 11 DRG-FRG genes stands out as a promising prognostic biomarker for HCC. Beyond its predictive value, it sheds light on the intricate crosstalk between DRG-FRG genes and HCC. Importantly, these findings could pave the way for enhanced prognostic prediction, informed treatment decisions, and the advancement of immunotherapy for HCC patients.

Association between awareness of harmful effects of smoking and smoking cessation advice provided by hospital chest physicians in Guangzhou, China: a multi-institutional cross-sectional survey.

BACKGROUND AND OBJECTIVE: It is unknown which factors are associated with smoking cessation interventions initiated by hospital chest physicians in China. We examined physicians' awareness of negative effects of smoking on smoking cessation advice given. METHODS: A cluster randomized cross-sectional survey was conducted from July to August 2011 in hospital chest physicians (n = 354) in Guangzhou, China. RESULTS: Of those who responded (n = 354, 92%), 63.8% were aware of negative effects of smoking and 64.5% initiated smoking cessation programmes with their patients. Awareness differed among physicians depending on their hospital affiliation (χ(2) = 54.7, P > 0.001), that is, primary (44.9%), secondary (55.1%) and tertiary hospitals (87.0%), (odds ratio = 1.732, 95% confidence interval: 1.072-2.797, P < 0.05). Physicians with less awareness prescribed smoking cessation medication less frequently (χ(2) = 137.71, P < 0.001). Smoking cessation practice by physicians also depended on hospital affiliation (χ(2) = 5.7, P > 0.001), (odds ratio = 4.074, 95% confidence interval: 1.399-11.860, P = 0.010). Smoking status of physicians was related to smoking cessation practice. CONCLUSIONS: Physicians more aware of the health hazards of smoking provided more smoking cessation advice. Awareness correlated with hospital levels and smoking status. Physician's advice correlated with their smoking status and educational background, but not with the levels of hospital, position or department affiliation.

d-mannose-sensitive pilus of Acinetobacter baumannii is linked to biofilm formation and adherence onto respiratory tract epithelial cells.

BACKGROUND/PURPOSE: Acinetobacter baumannii is an important nosocomial pathogen. To better understand the role of CsuA/BABCDE pilus of A. baumannii in virulence, bacterial biofilm formation, adherence and carbohydrate-mediated inhibition were conducted. METHODS: CsuA/BABCDE pilus-producing (abbreviated Csu pilus) operon of A. baumannii ATCC17978 was cloned for analysis of biofilm formation on an abiotic plastic plate, bacterial adherence to respiratory epithelial human A549 cells and carbohydrate-mediated inhibition. The carbohydrates used for inhibition of biofilm formation and adherence to A549 cells included monosaccharides, pyranosides, and mannose-polymers. RESULTS: The Csu pilus of A. baumannii ATCC17978 was cloned and expressed into a non-pilus-producing Escherichia coli JM109, and was knocked out as well. The recombinant Csu (rCsu) pilus on E. coli JM109/rCsu pilus-producing clone observed by both electro-microscopy and atomic force microscopy showed abundant, while Csu-knockout A. baumannii ATCC17978 mutant appeared less or no pilus production. The E. coli JM109/rCsu pilus-producing clone significantly increased biofilm formation and adherence to A549 cells; however, the Csu-knockout mutant dramatically lost biofilm-making ability but, in contrast, increased adherence. Moreover, both of biofilm formation and adherence could be significantly inhibited by d-mannose and methyl-α-d-mannopyranoside in Csu pilus-producing E. coli JM109, whereas in A. baumannii ATCC17978, high concentration of carbohydrates was required for the inhibition, suggesting that Csu pilus is sensitive to d-mannose. CONCLUSION: This is the first study confirming that Csu pilus of A. baumannii belongs to mannose-sensitive type 1 pilus family and contributes to biofilm formation and bacterial adherence to human epithelial cells.

Characterization of Salmonella resistance to bile during biofilm formation.

BACKGROUND: Non-typhoid Salmonella infection may present as acute gastroenteritis or chronic infection, primarily in the bile-rich gallbladder. Biofilm formation is a mechanism of bile resistance in Salmonella. Our aim was to determine how Salmonella utilizes bile as a signal, and to study the relevance of the interaction between the PhoP-PhoQ two-component system and cyclic diguanosine monophosphate (c-di-GMP) signaling to biofilm formation. METHODS: Two-dimensional (2-D) gel electrophoresis was used to identify genes required for Salmonella biofilm formation in bile. Quantitative real-time PCR (qRT-PCR) was used to clarify the role of the PhoP-PhoQ two-component system and its interaction with genes involved in the c-di-GMP network during biofilm formation. RESULTS: Our result revealed that Salmonella mutants with incomplete outer membrane (△ompA), defective flagella (△flgE), or incomplete PhoP-PhoQ two-component system (△phoP), were unable to develop complete biofilms in the presence of bile. Moreover, PhoP-PhoQ two-component system-related Salmonella mutants (△phoP, △phoQ, △phoP△phoQ) had lower expression of c-di-GMP related genes (csgD, adrA) than the wild-type Salmonella strain had in the bile environment. CONCLUSION: Salmonella may sense and respond to bile through the PhoP-PhoQ two-component system during biofilm formation. Furthermore, the PhoP-PhoQ two-component system might activate regulators of the c-di-GMP signaling network.

Application of next-generation sequencing to study ascitic microbiome in cirrhotic patients with or without spontaneous bacterial peritonitis.

BACKGROUND: Spontaneous bacterial peritonitis is an important cause of morbidity and mortality in cirrhotic patients with ascites. The key step in the pathogenesis of spontaneous bacterial peritonitis is bacterial translocation from intestinal lumen to mesenteric lymph nodes, and from there to the systemic circulation and ascitic fluid. We aimed to study the ascitic microbiota of cirrhotic patients with or without spontaneous bacterial peritonitis. METHODS: Both the 16S polymerase chain reaction approach and the whole genome shotgun approach were adopted for the next-generation sequencing technology. We compared the results derived from the two methods. RESULTS: The bacterial culture-negative ascites in cirrhotic patients, which even failed for amplification of 16S ribosomal DNA, were found to contain much less bacterial DNA than the culture-positive ones, indicating that the paucity of bacteria, instead of the difficulty of bacterial culture, was possibly the main reason for negative culture result of the ascites. Escherichia coli was the predominant species in all samples, and the bacteria of low abundance were also identified by the next-generation sequencing technology. CONCLUSION: Whole genome shotgun-based next-generation sequencing is an appropriate method for depicting the microbiome of ascites or of other specimens with a low abundance of bacterial DNA.

Carbapenem-resistant-only Pseudomonas aeruginosa infection in patients formerly infected by carbapenem-susceptible strains.

Pseudomonas aeruginosa isolates that were initially carbapenem-susceptible and later became selective carbapenem-resistant following antimicrobial therapy were identified from individual cases during the same hospitalisation. Cross-resistance to other ß-lactams was not found and their susceptibilities remained identical in consecutive isolates. Real-time quantitative reverse transcription PCR was performed to investigate the role of OprD, an outer membrane protein regulating the entry of carbapenems, in the appearance of carbapenem-resistant-only P. aeruginosa (CROPA). Fifteen paired isolates of carbapenem-susceptible P. aeruginosa (CS-PA) and CROPA were identified. All of the cases had carbapenem exposure history within 1 month before the appearance of CROPA (mean 10 days). Reduced OprD expression was found in 93% (14/15) of the isolates, suggesting that oprD inactivation was the major contributor to selective carbapenem resistance. Of the 14 cases with CROPA due to oprD mutation, 71% (10/14) were persistent infection, as genotype analysis revealed that their paired strains were isogenic; 29% (4/14) represented re-infections as they were heterogenic, suggesting that oprD-deficient CROPA existed in the hospital and that carbapenem selective pressure promoted its spread to patients. We conclude that CROPA may occur soon after the use of carbapenems to treat CS-PA infections and that oprD mutation is the major mechanism of resistance in CROPA. Restriction of empirical use of carbapenems by antibiotic stewardship is important to halt the occurrence of CROPA.

Use of twitch mouth pressure to assess diaphragm strength in patients with chronic obstructive pulmonary disease.

This study was undertaken to determine whether twitch mouth pressure (TwPmo) can reliably assess diaphragm strength in patients with chronic obstructive pulmonary disease (COPD) using fully automatic trigger techniques. Fifteen patients with COPD were recruited. TwPmo, twitch oesophageal pressure (TwPes) and twitch transdiaphragmtic pressure (TwPdi) were generated by phrenic nerve stimulation and were measured using an inspiratory flow trigger (40 ml/s, Experiment 1) using an inspiratory pressure trigger (-5 cmH2O, Experiment 2) and using no trigger at functional residual capacity (Experiment 3). The correlation between TwPmo and TwPes was as follows: r=0.832; P<0.0001 (Experiment 1), r=0.900; P<0.0001 (Experiment 2); there was no significant correlation in Experiment 3. A Bland-Altman plot of the difference between TwPmo and TwPes showed the limits of agreement in Experiment (1) bias (range) 0.18 cmH2O (-2.05 to 2.41) and Experiment (2) bias (range) 0.32 cmH2O (-1.69 to 2.32). Measuring TwPmo using a fully automatic technique is a simple and convenient method for assessing diaphragm strength.

[Association between awareness of smoking-related evidence and smoking cessation treatments provided by hospital chest physicians in Guangzhou, China: a multi-institutional cross-sectional survey].

OBJECTIVE: Factors associated with smoking cessation interventions initiated by hospital chest physicians in China had not been studied. We examined if the physicians' awareness of emerging evidence regarding negative effects of smoking was associated with the initiation of smoking cessation. METHODS: A cluster randomized cross-sectional survey was conducted from July 2011 to August 2011 in hospital-based chest physicians (n = 354) in Guangzhou, China. RESULTS: Of those who responded (n = 354, 92.2%), 63.8% were aware of emerging evidence regarding negative effects of smoking and 64.5% initiated smoking cessation programs with their patients who smoked. Regarding the related awareness on smoking differed across physicians depending on their affiliation to evidence hospitals(χ(2) = 54.7, P > 0.001), i.e., primary (44.9%), secondary (55.1%)and tertiary hospitals (87.0%)was further supported by the related odds ratio (OR = 1.732, 95%CI:1.072-2.797, P < 0.05). Smoking status of physicians was related to their practice on smoking cessation, supported by the odds ratio (OR = 4.251, 95% CI:1.460-12.380, P = 0.008). Smoking cessation practice by physicians also depended on their affiliated hospitals. Physicians working at primary and secondary hospitals were less aware of the fact that smoking could reduce patients' responsiveness to inhaled corticosteroids than those working at the tertiary hospitals(χ(2) = 37.9, P > 0.001). Furthermore, these physicians would less frequently prescribed medication related to smoking cessation(χ(2) = 137.71, P < 0.001). CONCLUSION: Physicians who were better aware of the health hazards of smoking might more actively provide smoking cessation advice in their clinics. The awareness might correlate with the hospital levels they worked and the smoking status while the the advice they provided might correlate with their educational background, job title, department affiliation and smoking status, but not with the level of hospitals.